Intervening sequences are spliced from eukaryotic precursor-tRNAs through the action of three enzymes, a heterotrimeric endonuclease, a multi-functional but monomeric ligase, and an NAD-linked phosphatase. The endonuclease and ligase form a physical complex that is localized in the nuclear periphery in the vicinity of nuclear pores, suggesting the potential for functional coupling of splicing and nuclear export. Two genes, SEN1 and SEN2, were identified previously in the yeast Saccharomyces cerevisiae that are required for endonuclease activity. SEN1 codes for a nuclear-localized, positive effector of endonuclease activity that appears not to be a catalytic subunit of the enzyme. SEN2 codes for the 42 Kd catalytic subunit of the enzyme. We have isolated mutations in six new genes that affect SEN1 function. Mutations in two of the genes, SEN3 and SEN4, affect the expression of SEN1 at a post-translational level. One temperature-sensitive mutation, sen3-l, also causes excess accumulation of tRNA splicing intermediates in vivo. In addition, four genes have been identified that suppress the temperature-sensitive senl-l mutation when present in multiple copies and may encode products that interact with the SEN1 protein. By continuing with our analysis of SEN1 and SEN2 function and by initiating analyses of SEN3, SEN4, and the high-copy suppressor genes, we hope to provide critical information on the structure, organization, function, and intracellular location of the splicing complex. It is also possible that we may learn something about the import of splicing enzymes into the nucleus and the export of spliced tRNA products from the nucleus.